Unraveling the Roles of Neuropeptides in the Chemosensation of the Root-Knot Nematode Meloidogyne javanica

The identification of novel drug targets in plant-parasitic nematodes (PPNs) is imperative due to the loss of traditional nematicides and a lack of replacements. Chemosensation, which is pivotal for PPNs in locating host roots, has become a focus in nematode behavioral research. However, its underlying molecular basis is still indistinct in such a diverse group of PPNs. To characterize genes participating in chemosensation in the Javanese root-knot nematode Meloidogyne javanica, RNA-sequencing of the second-stage juveniles (J2s) treated with tomato root exudate (TRE) for 1 h and 6 h was performed. Genes related to chemosensation in M. javanica mainly responded to TRE treatment at 1 h. Moreover, a gene ontology (GO) analysis underscored the significance of the neuropeptide G protein-coupled receptor signaling pathway. Consequently, the repertoire of putative neuropeptides in M. javanica, including FMRFamide-like peptides (FLPs), insulin-like peptides (ILPs), and neuropeptide-like peptides (NLPs), were outlined based on a homology analysis. The gene Mjflp-14a, harboring two neuropeptides, was significantly up-regulated at 1 h TRE treatment. Through peptide synthesis and J2 treatment, one of the two neuropeptides (MjFLP-14-2) was proven to influence the J2 chemotaxis towards tomato root tips. Overall, our study reinforces the potential of nematode neuropeptides as novel targets and tools for root-knot nematode control.


Introduction
Plant-parasitic nematodes (PPNs) pose a formidable threat to global agriculture, causing substantial yield losses in crops [1,2].For decades, nematicides have served as one of the primary interventions for controlling PPNs.However, the extensive restriction on nematicides' application and the emergence of drug resistance among nematode populations have exacerbated the impact of nematode infestations.Therefore, there is a growing recognition of searching for alternative approaches to nematode management.Biocontrol methods that exploit nematophagous fungi and bacteria and crop rotation with non-host plants represent environmentally friendly options by disturbing the life cycle and living environment of nematodes [3][4][5][6][7].Developing crop varieties carrying natural resistance genes via crop breeding or a genetic engineering approach is also an effective strategy [8,9].However, there are shortcomings associated with these strategies, such as economic losses, low application scope, unstable resistance, and resistance breakthroughs [10,11].Hence, it is necessary to develop novel management approaches.Considering the special neuronal uptake system of amphids in nematodes and its function in chemosensation, the disruption of nematode behaviors using neuropeptides as targets has become an innovative route for PPN control [12,13].
Neuropeptides are one of the most ancient signaling molecules and are abundant in the phylum Nematoda [14].In the model nematode Caenorhabditis elegans, there are at least 113 identified neuropeptide genes encoding over 250 putative neuropeptides [15].Nematode neuropeptides are classified into three large groups, the FMRFamide-like peptides (FLPs), the insulin-like peptides (ILPs), and the neuropeptide-like peptides (NLPs) [15].After being processed from precursors and post-translationally modified, bioactive neuropeptides are secreted from the nervous system and bind to their specific receptors on the cell surface [15].Besides ILPs, which signal through receptor tyrosine kinases, the majority of FLPs and NLPs are thought to be ligands of G-protein coupled receptors (GPCRs) [16,17].Coupled with the intricate and interconnected nature of cellular signaling pathways, neuropeptide-receptor couples have been proven to modulate various physiological processes in nematodes, such as chemosensation, locomotion, feeding, and reproduction [18,19].
Benefiting from the high conservation of neuropeptides within the phylum Nematoda and the implementation of the genome sequencing of PPNs, neuropeptide genes have been systematically analyzed and identified in several important PPNs, such as Globodera spp.and Meloidogyne spp.[20][21][22].In order to link neuropeptides to the coordination of nematode behaviors, especially for successful parasitism in plant roots, functional studies of those identified neuropeptides were carried out primarily using RNA interference technologies.For example, the silencing of flp-1, -6, -12, -14, or -18 in G. pallida resulted in abnormal locomotion phenotypes [23].After soaking with the double-stranded RNA (dsRNA) of M. incognita flp-14 and flp-18, the number of nematodes attracted by the host was significantly decreased, thereby resulting in a reduced nematode infection [24].The knockdown of nlp-3 and nlp-12 in M. incognita showed a significant reduction in attraction and infection in tomato roots [25].In addition, the use of synthetic bioactive neuropeptides is also an effective approach, with which a certain number of NLPs that impact chemosensation and host invasion was identified in M. incognita and G. pallida [26].However, as mentioned above, the studies of neuropeptides in PPNs are currently conducted in limited species, while the information of neuropeptides in other important PPNs is still lacking.
The Javanese root-knot nematode Meloidogyne javanica, as one of the most concerned root-knot nematodes, is widely distributed in warm and tropical regions [2].It establishes a permanent feeding site within the roots of a wide variety of host plants, including economically important crops.Here, we summarized the neuropeptide repertoire in M. javanica according to the annotation in the Wormbase Parasite database and performed homology-based BLAST to identify novel and missing entries that are not fully annotated.Based on the previous finding that tomato root exudate (TRE) was highly attractive to M. javanica J2s [27], the RNA-seq of M. javanica J2s treated with TRE was conducted, and the involvement of Mjflp-14 genes in regulating chemosensation in M. javanica emerged and was further confirmed using synthetic neuropeptides.

TRE Treatment Causes Transcriptional Changes in M. javanica
To explore how M. javanica responds to tomato root exudate (TRE) treatment at the transcriptome level, 12 libraries were constructed for RNA-seq, including J2s of M. javanica with 1 h and 6 h mock treatments and treatments with TRE.A total of 415,047,795 raw paired reads from 12 libraries was generated, of which 409,408,914 reads survived after filtering low-quality reads and adaptors (Table 1).Filtered reads from each library were aligned to the genome of M. javanica (ASM90000394v1) using HiSAT2 with an approximate mapping rate of 94.50-96.32%(Table 1).The PCoA analysis showed independent clustering between mock and TRE treatments (Figure 1A).The R package edgeR was used to further identify the differentially expressed genes (DEGs) in the comparison groups of mock1h_TRE1h and mock6h_TRE6h.A total of 1149 DEGs were identified in the TRE1h compared to the mock1h, of which 817 and 332 genes were up-regulated and down-regulated, respectively (Figure 1B, Table S1).Few numbers of DEGs were found in the mock6h_TRE6h comparison group, with 529 genes up-regulated and 246 genes down-regulated (Figure 1C, Table S1).Only 210 up-regulated and 54 down-regulated genes were shared between the 1 h and 6 h DEGs (Figure 1D,E).

M. javanica Primarily Responds to TRE at 1 h
To better understand the functional classification of DEGs, especially the genes positively responding to TRE treatment, the 20 genes with the highest up-regulated expression in 1h_up and 6h_up along with their gene function description were listed in Tables 2 and 3.Only 8 and 7 genes have a functional description in 1h_up and 6h_up, respectively.Notably, there were 3 genes, M.Javanica_Scaff2813g025362, M.Javanica_Scaff28572g095259, and M.Javanica_Scaff4717g035993, that were annotated as G protein-coupled receptors (GPCRs) and present only in 1h_up, and their expression levels have dropped dramatically at 6 h (Table S1).All the up-regulated DEGs in 1h_up and 6h_up were used for GO enrichment, respectively.A total of 32 GO terms were enriched in 1h_up, while only 10 GO terms were enriched in 6h_up (Table S2).Further, the top 10 GO terms selected by gene ratio were shown if the total number of GO terms belonging to molecular function (MF), cellular component (CC), or biological process (BP) exceeded 10 (Figure 2A,B).As shown in the results, a certain number of genes in 1h_up belongs to the GO terms related to chemical sensing and cellular signal transduction, including G-protein beta/gamma-subunit complex binding in MF, voltage-gated potassium channel complex in CC, and the sensory perception of the chemical stimulus in BP (Figure 2A, Table S2).Likewise, these GO terms were only found in 1h_up, while no such GO terms involved in these processes were enriched in 6h_up (Figure 2B).Taken together, M. javanica mainly responds to TRE more actively at transcriptome levels at the earlier stage (1 h) than the later stage (6 h) treatment.mock6h_TRE6h comparison group, with 529 genes up-regulated and 246 genes downregulated (Figure 1C, Table S1).Only 210 up-regulated and 54 down-regulated genes were shared between the 1 h and 6 h DEGs (Figure 1D,E).GPCRs mediate responses to various extracellular and intracellular cues and represent the largest family of cell surface proteins in metazoan [28,29].However, the functional overlap among GPCRs poses a challenge for the dissection of their functions.As GPCRs were found to regulate chemosensation in C. elegans as receptors of neuropeptides [30], it prompted us to consider whether neuropeptide genes in M. javanica also respond to TRE.Due to the lack of a summary of neuropeptides in M. javanica, the first attempt was made to identify genes encoding neuropeptides at the genome level.Through sequence alignment and a consensus analysis, a total of 74 neuropeptide genes were obtained in M. javanica, including 23 FMRFamide-like peptides (flps), 20 insulin-like peptides (ilps), and 31 other neuropeptide-like proteins (nlps) (Figure 3A, Table S3).All of the genes identified except ilps were successfully classified and named in accordance with the consensus peptide sequences of known neuropeptides in C. elegans (Table S3).Although these three types of genes were not fully discriminative clustering in the phylogenetic analysis, the paralogous genes were strictly clustered into the same branch (Figure 3B).Combining with the RNAseq data, 3 of the 74 neuropeptide genes were found to be significantly up-regulated under TRE treatment.The gene M.Javanica_Scaff1293g014508 (Mjflp-14a) was significantly up-regulated at 1 h of TRE treatment, and two ilp genes M.Javanica_Scaff33g000778 and M.Javanica_Scaff2132g020889 were up-regulated both at 1 h and 6 h (Figure 3C).

The Precursors of MjFLP-14 Neuropeptides (Pro-Neuropeptide) Contain Two Neuropeptides
The pro-neuropeptide sequences of the three responsive genes were further analyzed using the homologs of C. elegans as a reference to determine the processed neuropeptides.However, no homologs of the two ILPs encoding M.Javanica_Scaff33g000778 and M.Javanica_Scaff2132g020889 were found in C. elegans.The four MjFLP-14 pro-neuropeptides showed sequence similarities with the FLP-14 in C. elegans, with the highest identity of MjFLP-14b reaching to 62.16% (Figure 4A).A further comparison of the FMRFamide-like domain revealed that all MjFLP-14 had two distinctly processed neuropeptides, MjFLP-14-1 (KHEYLRFG) and MjFLP-14-2 (KHEFVRFG), flanked by dibasic residues (Figure 4B).MjFLP-14-1, which shared the same sequence as the neuropeptide in CeFLP-14, was located in the front, followed by MjFLP-14-2, which has two residues different from MjFLP-14-1 (Figure 4B).In addition, unlike CeFLP-14 possessing four copies of the neuropeptides,  were displayed in red, green, and blue, respectively.GO terms related to cell signaling were bolded.

MjFLP-14-2 Affects the Root Chemotaxis of M. javanica
The increased gene expression of Mjflp-14a at TRE1h was further confirmed with RT-qPCR (Figure 5A).To test whether MjFLP-14-1 and MjFLP-14-2 affect nematode behaviors, the two neuropeptides were synthesized with the functional post-translational modification of C-terminal amidation (Figure 5B).The effect of bioactive peptides on the root chemotaxis of M. javanica J2s was determined.The results showed that after being treated with MjFLP-14-2 at final concentrations of 50 and 100 µM for 12 and 24 h, the number of J2s attracted by root tips was significantly lower than that of the mock treatment, but no significant difference was found at 20 µM (Figure 5C).There is no significant difference between the mock and MjFLP-14-1 treatments (Figure 5C).
OR PEER REVIEW 7 of 14 The pro-neuropeptide sequences of the three responsive genes were further analyzed

MjFLP-14-2 Affects the Root Chemotaxis of M. javanica
The increased gene expression of Mjflp-14a at TRE1h was further confirmed with RT-qPCR (Figure 5A).To test whether MjFLP-14-1 and MjFLP-14-2 affect nematode behaviors the two neuropeptides were synthesized with the functional post-translational modification of C-terminal amidation (Figure 5B).The effect of bioactive peptides on the root chemotaxis of M. javanica J2s was determined.The results showed that after being treated with MjFLP-14-2 at final concentrations of 50 and 100 μM for 12 and 24 h, the number of J2s attracted by root tips was significantly lower than that of the mock treatment, but no significant difference was found at 20 μM (Figure 5C).There is no significant difference between the mock and MjFLP-14-1 treatments (Figure 5C).

MjFLP-14-2 Affects the Root Chemotaxis of M. javanica
The increased gene expression of Mjflp-14a at TRE1h was further confirmed with RT-qPCR (Figure 5A).To test whether MjFLP-14-1 and MjFLP-14-2 affect nematode behaviors, the two neuropeptides were synthesized with the functional post-translational modification of C-terminal amidation (Figure 5B).The effect of bioactive peptides on the root chemotaxis of M. javanica J2s was determined.The results showed that after being treated with MjFLP-14-2 at final concentrations of 50 and 100 μM for 12 and 24 h, the number of J2s attracted by root tips was significantly lower than that of the mock treatment, but no significant difference was found at 20 μM (Figure 5C).There is no significant difference between the mock and MjFLP-14-1 treatments (Figure 5C).

Discussion
The successful parasitism of PPNs in plant roots requires a series of behavioral coordinations, including chemosensation, locomotion, penetration, and feeding.Among these behaviors, chemosensation represents an early step in the process of host finding.Chemicals in root exudates secreted by host plants act as signals for root-knot nematodes, attracting them towards the root tip region of host plants [27,[31][32][33][34]. Therefore, understanding the interplay between root exudates and PPNs is crucial for developing strategies to manage nematodes.We designed an experiment to treat M. javanica with collected tomato root exudates alone and then used them for RNA-seq, by which it is possible to exclude the effects of physical interaction with plant roots and focus on how the root exudates affect nematodes.The feasibility of this treatment was confirmed with a GO enrichment analysis of the DEGs.There are GO terms related to signal sensing and transduction pathways enriched in the 1h_up as well as pectate lyase activity, which is found both in the 1h_up and 6h_up (Figure 2A,B).These results indicate that treatment with tomato root exudate alone can successfully induce a response in nematodes, as it does around the roots.
Neuropeptides and components of the signal pathways where they are involved have become intriguing targets for PPN control [12].The present study identified a total of 74 neuropeptide genes in M. javanica, whose genome is proposed to be tetraploid [21].Indeed, we found that many groups of neuropeptides possess four paralogous genes; however, a certain number of groups had only three paralogous genes (Figure 3B).This may be due to loci loss during polyploidization or incomplete sequence assembly and annotation.The FLPs and NLPs in M. javanica showed high sequence conservation and were effortlessly assigned into the same groups as in C. elegans.However, although the insulin-like domain was present in all ILPs in M. javanica, no homologs of C. elegans were found, even for the INS-1, -17, and -18 that are found to be widely distributed within nematodes, including plant-parasitic, human-parasitic, animal-parasitic, EPN, and freeliving nematodes [35].Therefore, they were not assigned and named by homology.
It is not yet known how fast the nematodes respond to TRE after sensing it, so two time points (1 h and 6 h) of mock and TRE treatments were chosen for the RNA-seq.In the DEGs list of RNA-seq, only 3 of 74 neuropeptide genes, Mjflp-14a and two Mjilp genes, were included.This is explainable because PPNs are able to perceive root exudates and thereby tailor gene expression for what is currently needed for host parasitism [36].The two ilp genes exhibited a similar expression pattern, as they are paralogs.However, of the four Mjflp-14 genes, only Mjflp-14a was significantly up-regulated.The contingency derived from the experiment or RNA sequencing was first excluded because the abundance of Mjflp-14a gene expression has increased dramatically at 1 h of TRE treatment (Figure 3C).A possible reason is that the expression of Mjflp-14a is under a special regulatory mechanism in the process of perceiving the host.In addition to comparing the mock1h_TRE1h and mock6h_TRE6h groups, the DEG analysis was also performed between mock1h and mock6h (mock1h_mock6h).Only 41 and 113 genes were significantly up-regulated and down-regulated, respectively.No specific GO terms of biological processes were enriched within these DEGs, suggesting a stable status of J2s in the short time scale.Moreover, the consistent expression level of Mjflp-14a in both mock1h and mock6h suggests a stable expression of the gene in the absence of TRE.
FLP-14 is a highly conserved and abundant neuropeptide amongst nematodes [37,38].It was found to be expressed in neurons of C. elegans and the SMB-like neurons of the PPN M. incognita, implying its role in regulating locomotion and sensory processing [38,39].In PPNs, the significance of flp-14 for the infection and development of M. incognita was demonstrated through the RNAi of the gene in vitro and in planta [24,40].The similar phenotype of flp-14 silencing was also found in M. graminicola [41].These studies demonstrated a deficient expression level of flp-14-affected nematode behaviors.Oppositely, our study used synthetic MjFLP-14-2 to treat nematodes, which is equivalent to excessive levels and also obtained similar results of negatively regulating chemotaxis.This can be explained by the precise regulatory requirement of flp-14 levels in C. elegans, pointing out that abnormal levels of flp-14 and its receptor of the same neuropeptide pathway, whether excessive or deficient, disrupt normal functions, leading to significant behavioral defects [39].Thus, our study indicated a conserved function of flp-14 amongst nematodes.Unlike the FLP-14 in C. elegans, which was processed into one active neuropeptide, MjFLP-14 is able to be processed into two similar but different neuropeptides (Figure 4B).Nevertheless, only MjFLP-14-2 showed negatively regulation in chemotaxis of M. javanica via treating the J2s with a synthetic peptide.Unlike MjFLP-14-1, the MjFLP-14-2 peptide sequence is slightly different from FLP-14 in C. elegans, indicating variations of activity or the function of similar neuropeptides among distinct nematode species.The role of MjFLP-14-2 in negatively affecting the host finding of M. javanica and potentially other RKNs could designate it as a promising target for interference strategies aimed at preventing RKN infections in crops.

Nematode Propagation
The root-knot nematode Meloidogyne javanica was cultured on tomato plants (cv.Rutgers) under greenhouse conditions (23-26 • C, 16/8 h day/night period).Eggs of M. javanica were collected by treating the infected tomato roots with 0.8% NaClO for 5 min followed by continuous washing through a series of 250, 45, 25 µm mesh sieves.Freshly prepared eggs were used for the second-stage juveniles (J2s) hatching in 0.1% (v/v) plant preservative mixture (PPM) solution.

Tomato Root Exudate (TRE) Collection
TRE was collected as described with modifications [27].Rutgers tomato seeds were germinated on autoclaved germination paper soaked in autoclaved deionized water (DiH 2 O) in petri dish for 10 days.An amount of 70 seedlings were rolled together such that the terminal 10 mm of roots were submerged in 8 mL of autoclaved DiH 2 O in a 100 mL beaker.The 100 mL beaker was placed in a 500 mL beaker with a wet Kimwipe ® tissue (Kimtech Science™, Dallas, TX, USA) at the bottom, and the whole setup was covered with a sheet parafilm with a few pin holes for ventilation.The setup was kept at 25 • C in the dark.After 24 h, the liquid in the 100 mL beaker was collected and passed through a 0.22 µm filter.This TRE can be used immediately or stored at −20 • C.

Sample Collection for RNA-Seq
Freshly hatched M. javanica J2s were concentrated by natural settling down for 1 h, and the concentration of J2s was determined.An amount of 1.2 mL of TRE was added to each well of the 12-well plate, and then, 2000 J2s were added and mixed well.The plates were kept at 25 • C in the dark and gently shaken every 20 min.The solutions were transferred to 1.5 mL low-retention tubes after 1 h and 6 h and centrifuged at 7000 rpm for 1 min.The supernatant was carefully removed and then snap frozen in liquid nitrogen before being stored at -80 • C. Autoclaved DiH 2 O instead of TRE was used as mock treatment.

RNA Extraction, Library Preparation, and RNA-Seq
Total RNA of each sample was extracted using the PureLink ® RNA Mini Kit (Invit-rogen™, Waltham, MA, USA) following the manual with three technical replicates.The quality and quantity of RNA from each sample were determined by the Agilent 5400 system in Genomics Core at Purdue University, and the RNA integrity numbers (RINs) of all samples were larger than 9.

Analysis of RNA-Seq Data
Raw reads from each sample were trimmed to filter low-quality bases at both 5 ′and 3 ′ -ends, length below 50 bp, and the contaminated adapters using Trimmomatic 0.38 [42].The clean reads generated were mapped to the reference genome of M. javanica, downloaded from the Wormbase Parasite (https://parasite.wormbase.org/Meloidogyne_javanica_prjeb8714/Info/Index, accessed on 16 October 2023) using HiSAT2 software (v 2.2.1) [43].SAM files were converted into BAM files and sorted using Samtools-1.15.fea-tureCounts was employed to count mapped reads, taking exon as a feature [44].Differential gene expressions between control and treatment were determined on the counts using the Bioconductor software edgeR v 3.38.4[45].Genes were considered to be significant DEGs when the absolute value of log 2 (fold change) > 1 and the false discovery rate (FDR) < 0.05.The GO enrichment of DEGs was implemented using the enricher function provided by the clusterProfiler R package v 4.4.4[46].The GO annotation file of M. javanica was downloaded from BioMart in the Wormbase Parasite database and used for enrichment analysis.GO terms with adjusted p-value < 0.05 were considered significantly enriched GO terms.

BLAST Searches for Neuropeptide Genes in M. javanica
Two methods were combined to identify the neuropeptide genes in M. javanica.For the flp and nlp genes with known sequence homology between C. elegans and M. javanica [14,26,47], the propeptide sequences of FLPs and NLPs in C. elegans were successively used as queries to perform BLASTP search against the genome of M. javanica (PRJEB8714) on the Wormbase Parasite server (https://parasite.wormbase.org/Multi/Tools/Blast,accessed on 24 October 2023) with default setting.For the ilp genes lacking homology information, the function of display all genes with this domain on the Wormbase Parasite was used to obtain all genes harboring the domain of insulin-like superfamily (Inter-Pro, IPR036438) in M. javanica.The FMRFamide-related peptide-like domain (InterPro, IPR002544) was also applied to this function search to generate a new output of flp genes, which was merged with the previous output of flp genes after removing duplicates.Then, the resulting outputs of these three types of genes were used as queries again to perform BLASTP against the M. javanica genome to search for missing paralogous genes.Each group of paralogous genes was named alphabetically according to the last 6 digits of the gene ID from smallest to largest.To obtain the conservation of all neuropeptides in M. javanica with other nematodes, BLASTP search was performed using the amino acid sequences of neuropeptides to search against the genome of C. elegans (PRJEB13758), M. incognita (PRJEB8714), M. hapla (PRJEB29083), M. enterolobii (PRJEB36431), M. arenaria (PRJEB8714), M. chitwoodi (PRJNA666745), and Heterodera glycines (PRJNA381081).

Post-BLAST Analysis
All BLAST-generated genes were manually inspected for consensus peptide sequences flanked by mono/dibasic cleavage sites, and the sequences that showed sequence homology but did not contain consensus peptide sequences were excluded.The remaining genes were designated to the cluster of neuropeptides in C. elegans based on sequence homology.Signal peptides were predicted by SignalP 6.0 [48], but the sequences lacking a predicted signal peptide did not exclude neuropeptide designation.

Phylogenetic Tree Construction
Multiple sequence alignment of the neuropeptide genes in M. javanica was carried out using the MUSCLE program embedded in the MEGA 11 software (v 11.0.9) with default setting [49].The result of alignment was used as an input to construct the tree using the neighbor-joining method based on the poisson distribution.Interactive Tree of Life server (https://itol.embl.de/,7 December 2023) was employed to annotate the tree.

Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)
The total RNA was treated with the TURBO DNA-free™ Kit (Invitrogen™, Waltham, MA, USA) and used to generate the first strand of cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA).Gene expression abundance of Mjflp-14a was analyzed using the Bio-Rad CFX96 Real Time System using the primers (forward: 5 ′ -TCTTCTACACTTCTGTCTCAATTAGG-3 ′ and reverse: 5 ′ -AACGCAAATACTCATGCTTTCTC-3 ′ ).Gene GAPDH in M. javanica were used as endogenous reference genes using the primers (forward: 5 ′ -CGTGCAGCGGTTGAGAAGGA-3 ′ and reverse: 5 ′ -ACGTCCGTGGGTAGAATCAT-3 ′ ).The fold changes of gene expression were calculated versus the control by the 2 −△△Ct method.

Peptide Synthesis and Root Attraction Assay
Two predicted FLP-14 peptides in M. javanica were synthesized at GenScript (Piscataway, NJ, USA) with a modification of C-terminal amidation and dissolved in DMSO to make a 100 mM stock.Rutgers tomato seeds were germinated on autoclaved germination paper soaked in autoclaved deionized water (DiH 2 O) in petri dish for 7 days.On the 6th day of germination, freshly hatched J2s of M. javanica were incubated in 500 µL of each peptide solution at a final concentration of 20, 50, and 100 µM in 1.7 mL low-retention tubes for 12 and 24 h.An amount of 2 mL of 0.85% (w/v) water agar was added to each well of the 6-well plate, and then, 200 µL of 500 treated J2s was added and mixed well.One germinated seedling was placed in each well with the root tips submerged in the water agar.The plates were placed at 25 • C in the dark for 12 h, and the number of J2s attracted by root tips was counted.An equal volume of DMSO instead of peptide was used as mock treatment to go through the same steps.

Statistics Analysis
Data were presented as mean ± standard error of mean (SEM), and all statistical tests were performed using GraphPad Prism 10 (v 10.1.1,GraphPad Software, La Jolla, CA, USA).Comparisons between groups were performed using unpaired two-tailed parametric t test.A p-value < 0.05 was considered statistically significant.

Figure 1 .
Figure 1.The intergroup analysis of RNA-seq data.(A) The PCoA analysis of the samples in RNAseq.The fragments per kilobase million of each sample were used for drawing.(B,C) Volcano plots of differentially expressed genes (DEGs) in the comparison groups of mock1h_TRE1h (B) and mock6h_TRE6h (C).Genes with the false discovery rate < 0.05 were used to generate volcano plots, and the log2(fold change) > 1 and log2(fold change) < −1 were considered as up-regulated and downregulated DEGs, respectively.(D,E) Venn diagrams of DEGs between the 1h_up and 6h_up (D) and 1h_down and 6h_down (E) groups.

Figure 1 .
Figure 1.The intergroup analysis of RNA-seq data.(A) The PCoA analysis of the samples in RNAseq.The fragments per kilobase million of each sample were used for drawing.(B,C) Volcano plots of differentially expressed genes (DEGs) in the comparison groups of mock1h_TRE1h (B) and mock6h_TRE6h (C).Genes with the false discovery rate < 0.05 were used to generate volcano plots, and the log2(fold change) > 1 and log 2 (fold change) < −1 were considered as up-regulated and down-regulated DEGs, respectively.(D,E) Venn diagrams of DEGs between the 1h_up and 6h_up (D) and 1h_down and 6h_down (E) groups.

Figure 2 .
Figure 2. The gene ontology (GO) enrichment analysis of the DEGs in 1h_up (A) and 6h_up terms belonging to the biological process (BP), cellular component (CC), and molecular f (MF) were displayed in red, green, and blue, respectively.GO terms related to cell signalin bolded.

Figure 2 .
Figure 2. The gene ontology (GO) enrichment analysis of the DEGs in 1h_up (A) and 6h_up (B).GO terms belonging to the biological process (BP), cellular component (CC), and molecular function (MF) were displayed in red, green, and blue, respectively.GO terms related to cell signaling were bolded.

Figure 4 .
Figure 4.The sequence analysis of FLP-14 paralogs in M. javanica (MjFLP-14).(A) The sequence multiple alignment of MjFLP-14 with the FLP-14 in C. elegans (CeFLP-14).Residues were shaded with gradient colors according to the amino acid conservation.(B) Schematic diagram of the protein sequences of MjFLP-14 and CeFLP-14 in proportion to the length.

Figure 4 .
Figure 4.The sequence analysis of FLP-14 paralogs in M. javanica (MjFLP-14).(A) The sequence multiple alignment of MjFLP-14 with the FLP-14 in C. elegans (CeFLP-14).Residues were shaded with gradient colors according to the amino acid conservation.(B) Schematic diagram of the protein sequences of MjFLP-14 and CeFLP-14 in proportion to the length.

Figure 4 .
Figure 4.The sequence analysis of FLP-14 paralogs in M. javanica (MjFLP-14).(A) The sequence multiple alignment of MjFLP-14 with the FLP-14 in C. elegans (CeFLP-14).Residues were shaded with gradient colors according to the amino acid conservation.(B) Schematic diagram of the protein sequences of MjFLP-14 and CeFLP-14 in proportion to the length.

Table 2 .
The description of the top 20 up-regulated DEGs (mock1h_TRE1h).

Table 3 .
The description of the top 20 up-regulated DEGs (mock6h_TRE6h).